Tumor necrosis factor-a- (TNF) and mitochondrial GSH (mGSH) are two factors that modulate alcohol-induced liver injury and progression of alcoholic liver disease (ALD). The overall goal of this proprosal is to identify the signaling intermediates generated by TNF that target mitochondria and the role of mitochondrial cholesterol and lipid composition on the regulation of mGSH and mitochondrial permeability transition (MPT). Specific aims : 1. Role of acidic sphingomyelinase (ASMase) on alcohol-mediated hepatocellular injury. Ceramide functions as a lipid death effector through its interaction with mitochondria. Thus, we will examine the specific role of ASMase on the sensitization of the liver to alcohol feeding vs. known contributors of TNF cytotoxicity (Bid, cathepsin B). Rats with inactive endogenous ASMase and ASMase deficient mice will be fed ethanol chronically evaluating the sensitization of the liver to alcohol-induced liver injury. 2. Contribution of ganglioside GD3 to the progression of ALD. Ganglioside GD3 (GD3), a sialic-acid containing glycosphingolipid synthesized from ceramide within the Golgi network, interacts with mitochondria by an actin cytoskeleton vesicular trafficking. The intracellular trafficking of GD3 will be characterized in hepatocytes from alcohol-fed rats and its role in alcohol liver injury will be assessed in GD3 synthase knockout mice. 3. Regulation of mGSH by cholesterol: intramitochondrial cholesterol trafficking and role of peripheral benzodiazepine receptor (PBR). The mGSH arises by the mGSH carrier whose activity is sensitive to altered membrane fluidity due to increased cholesterol deposition. We will determine the temporal relationship between incrased mitochondrial cholesterol and mGSH depletion by alcohol feeding. In addition, we will examine the regulation of cholesterol by alcohol, the role of hydroxymethylglutaryI-CoA reductse and the collaboration of PBR in the intramitochondrial trafficking of cholesterol. 4. MPT: role of cardiolipin and MPT components. MPT is key in TNF-mediated hepatocellular injury and alcohol has been shown to potentiate MPT. Since cardiolipin, an exclusive mitochondrial phospholipid, has been recently suggested to interact with proapoptotic Bcl-2 family members that induce MPT,we will examine the role of cardiolipin vs MPT components reconstituted liposomes in MPT regulation in mitochondria and mitoplasts isolated from alcohol-fed rats.